Haemophilus influenzae B polysaccharide-diptheria toxoid conjugate vaccine

ABSTRACT

A water-soluble covalent polysaccharide-diphtheria toxoid conjugate having a molecular weight between 140,000 and 4,500,000 dalton and a ribose/protein ratio between 0.25 and 0.75, capable of producing T-cell dependent antibody response to polysaccharide from H. influenzae b, is prepared by mixing a derivatized diphtheria toxoid in a substantially cyanogen halide-free solution with a cyanogen halide-activated capsular H. influenzae b polysaccharide hapten consisting of approximately equal parts of ribose, ribitol and phosphate, which polysaccharide had previously been heat sized until more than 60% attained a molecular size between 200,000 and 2,000,000 dalton.

This application is a continuation of Ser. No. 395,743, filed July 6,1982 now U.S. Pat. No. 4,496,538.

BACKGROUND OF THE INVENTION

Haemophilus influenzae b is the most common cause of bacterialmeningitis and other invasive infections in children in the UnitedStates. Extrapolating from prospective data gathered in Fresno County,California, approximately one in 300 newborns in the United States willdevelop bacteritic H. influenzae disease before the age of five years,and two-thirds of these episodes are associated with meningitis. Despitetherapy with antibiotics, the mortality of Haemophilus meningitisremains at approximately 5%, and serious neurologic sequalae occur in asmany as 10 to 25% of survivors. Since 1974, strains of Haemophilusresistant to ampicillin have emerged in the United States. By 1981 thesestrains were prevalent in nearly all communities, and b Haemophilusstrains resistant to chloramphenicol or to multiple antibiotics havealso been described. Recently, type b Haemophilus has been implicated asa cause of epidemic disease in children attending day care centers, aproblem which appears to be increasing in dircet proportion to theincrease in the number of women entering the work force. These concernshave heightened interest in developing a vaccine for prevention ofsystemic Haemophilus infections in infants and young children.

Considerable data indicate that the type b capsule of Haemophilusinfluenzae, a heteropolymer of approximately equal amounts of ribitol,ribose and phosphate (PRP), is an important determinant of virulence,enabling the organism to resist host defenses. Antibodies directed atthe type b capsule confer protection against disease by initiatingopsonization and complement-dependent bacteriolysis. During the lastthree decades, considerable effort has been made to purify andcharacterize this capsular polysaccharide. A type b capsularpolysaccharide vaccine was prepared which was immunogenic in adults andin children older than 18 months of age, but failed to elicit immunityin infants, the age group at greatest risk of Haemophilus disease.(Makela, P. M. et al., J. Infect. Dis. 136: S43; 1977).

The purified type b polysaccharide appears to act as athymic-independent antigen. Booster immunization does not result ineither a memory or an accelerated antibody response (Anderson, P. etal., J. Infect. Dis. 136, S57; 1977).

The carrier-hapten concept was established by Landsteiner in 1926, and ageneral method used to create a covalent link between PRP and a proteincarrier was published by Schneerson et al. in 1980 (J. Exp. Med. 152,361; 1980). In such a vaccine an antigenic, but weakly ornon-immunogenic molecule, a hapten, contributes a new antigenicspecificity to an immunogenic carrier molecule.

SUMMARY OF THE INVENTION

The invention relates to a new Haemophilus influenzae bpolysaccharide-diphtheria toxoid conjugate (PRP-D) vaccine, thepolysaccharide hapten used for making same and the process for producingthem. More specifically the invention provides a pure Haemophilusinfluenzae b polysaccharide of a molecular size principally between200,000 and 2,000,000 dalton which is activated with cyanogen halide,typically the bromide. The activated polysaccharide is intimately mixedwith a toxoid, preferably diphtheria toxoid, to effect conjugation.Preferably, the toxoid is derivatized using as a spacer a bridge of upto 6 carbons, such as provided by use of the adipic acid hydrazidederivative of diphtheria toxoid (D-AH).

By use of this method there is obtained a conjugate vaccine whichelicits a T-cell dependent response to polysaccharide from Haemophilusinfluenzae b.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically illustrates the chromatographic profile for the bulkpolysaccharide-diphtheria toxoid conjugate (PRP-D) of Example IV.

FIG. 2 demonstrates the immunogenicity of Haemophilus influenzae bcapsular polysaccharide, i.e., the anti-PRP response. This bar graphpresents the data of Table 1, according to the protocol of Example V.

DISCLOSURE OF THE INVENTION

The development of stable humoral immunity requires the recognition offoreign material by at least two separate sets of lymphocytes. Thesesets are the B-lymphocytes, which are the precursors of antibody formingcells, and the T-lymphocytes which modulate the function of B-cells.While some antigens, including several polysaccarides, are capable ofdirectly stimulating B-cells to produce antibody (T-independentantigens), most antigens (T-dependent) must be presented to the B-cellby a T-lymphocyte. In the case of the vaccine of this invention, thediphtheria toxoid portion of the vaccine is recognized by the T-cellsystem. Since the protein carries both its own antigenic determinantsand the covalently bound PRP hapten, both sets of determinants should bepresented by T-cells to B-cells. The result of administration of thiscarrier-hapten preparation is that PRP is presented as a T-dependentimmunogen. There is good reason to expect that a T-dependentpresentation of PRP will induce protective immunity in infants, thetarget population at greatest risk.

T-independent antigens induce B-cells to terminally differentiate intoantibody secreting cells (plasma cells), while T-dependent responses areconsiderably more complex. After receiving a T-dependent stimulus, theB-cell population enters not only antibody production, but alsoproliferation and maturation. As a result, there should be an increasein the number of B-cells making antibodies to PRP and an increase in thenumber of B-cells capable of responding to a second exposure to PRP.Repeated immunization should result in further increases in the numberof PRP specific B-cells and, consequently, higher antibody titers, abooster response. In summary, while T-independent responses are limitedby the number of responsive B-cells, T-dependent responses result in anincrease in the total number of antigen responsive cells.

The vaccine of this invention, PRP-D, has been shown to function as aT-dependent immunogen in laboratory animals. Thirteen out of thirteenrabbits serially immunized with a standard dose of PRP-D showed boosterresponses. In addition, primary immunizations with the carrier protein,diphtheria toxoid, were shown to augment the initial response to the PRPcomponent of the PRP-D conjugate. A similar augmentation of the PRPresponse was not seen in animals primed with tetanus toxoid.

The vaccine of this invention is a PRP-D hapten-carrier conjugate. Insuch vaccines, the antigenic but weakly immunogenic hapten molecule(PRP) contributes a new antigenic specificity to a highly immumogeniccarrier molecule (D).

The purified diphtheria toxoid (D) used as carrier in the preparation isa commercial toxoid modified (derivatized) by the attachment of a spacermolecule, such as adipic acid dihydrazide (ADH), using the water-solublecarbodiimide condensation method. The modified toxoid, typically theadipic hydrazide derivative D-AH, is then freed from unreacted ADH.

Haemophilus influenzae b capsular polysaccharide is prepared fromcommercial sources such as used for licensed polysaccharide vaccines.However, while the polysaccharide is conventionally purified as acalcium salt, the use of calcium ions is avoided because they interferewith the use of carbonate buffer in the conjugation procedure. Themolecular size of the polysaccharide is then adjusted by heating untilthe desired dimension for the hapten is obtained. Typically, heating ofthe liquid polysaccharide for 15 minutes at 100° C. suffices to assurethat less than 20% of the molecules are of a molecular size smaller than200,000 dalton and less than 20% of a molecular size greater than2,000,000 dalton. This sizing operation is important to obtain a properPRP-D from unreacted protein.

The sized polysaccharide thus obtained is activated with cyanogenhalide, typically bromide, to create an electrophilic group on thepolysaccharide. Unreacted cyanogen halide is exhaustively removedbecause, if there is a substantial residue, it causes cross-linking ofthe protein in the following reaction mixture. The resultingcross-linked product traps polysaccharide, producing a higher molecularweight conjugate which is different in chemical properties from theconjugate produced herein and a vaccine which lacks the desirableproportion of the vaccine of this invention.

The activated PRP and D-AH are then combined and allowed to react in thecold. Some of the hydrazide groups on the derivatized diphtheria toxoidreact with the activated sites on PRP to form covalent bonds. Theproduct is PRP covalently bound to derivatized D through a six carbonchain. This reaction product is purified by gel permeationchromatography to remove any unreacted protein and low molecular weightcontaminants of molecular weight lower than 140,000 dalton. The typicalmolecular weight of the principal fraction is about 675,000 daltonrelative to dextran standard. A typical range for relative molecularsize is 140,000 dalton to 4,500,000 dalton.

A preservative such as thiomerosal is added, in the case of thiomerosal,to a final concentration of 1:10,000. The bulk concentrate (PRP-D) isfiltered through a 0.2 micron Durapore membrane and stored in the cold.

The following examples are provided for purposes of illustrating theinvention in further detail. They are not to be construed as limitingthe invention in spirit or in scope.

EXAMPLE I Preparation of PRP Used

A. Organism

Capsular polyribosyl ribitol phosphate (PRP) of Haemophilus influenzae bwas prepared from the commercially used Eagan strain. The culture wasrepeatedly transferred and a lyophilized seed prepared. From thislyophilized seed one additional transfer was made to prepare wet workingseeds (stored at -60° C.). Fermenter lots of bacterial cells wereprepared from the wet working seed.

Culture purity is determined by the following criteria:

1. negative Gram stain characteristics;

2. growth on agar containing NAD (diphosphopyridine nucleotide) andHemin (Bovine Type I crystalline salt of ferriheme);

3. failure to grow on agar without NAD or Hemin; and

4. agglutination by specific antisera (type b Haemophilus influenzae b,Hyland Laboratories).

B. Cultivation and Media

For subculturing the bacterium, i.e., preceding inoculation of afermenter, BHI Agar (per liter: 37 gm BHI Difco, 15 gm Bacto Agar Difco,0.6 ml 1% NAD Sigma-Grade III and 6 ml of 0.2% Hemin (Sigma-BovineType 1) was employed. Cells (20 hours) washed from an agar surface areused to inoculate Haemophilus influenzae b (Hib) liquid media (1 literaliquots); these cultures are incubated with shaking until the bacteriumreaches the log phase of its growth cycle. At this time, 2 liters ofculture are used to inoculate each 40 liters of liquid Hib media in afermenter and 300 ml 8% UCON (Union Carbide lubricant) is added. After16 to 18 hours, the fermenter culture is ready for harvest.

The composition of the Hib liquid media per 1000 ml is:

    ______________________________________                                        Yeast Extract Dialysate, Difco                                                                      5.0        gm                                           Casamino Acids, Difco 22.5       gm                                           Sodium Phosphate, dibasic                                                                           14.4       gm                                           Dextrose              5.59       gm                                           Hemin                 20         gm                                           Ammonium Hydroxide (30%)                                                                            0.1534     ml                                           NAD 1%                0.6        ml                                           ______________________________________                                    

When harvesting a fermenter, culture purity is determined by appropriateGram staining and culturing techniques (see above). Cetavlon(hexadecyltrimethylammonium bromide) is added to the culture to a finalconcentration of 0.1%. After 30 minutes, at which time the bacteria havebeen inactivated, the solid paste is collected by centrifugation. Thewet paste is stored at -70° C. until further processing.

C. Purified Polysaccharide

The extraction and subsequent purification of the PRP is carried outusing the following procedure:

1. Dissociation from Detergent

For each gram wet weight of paste, 10 ml of 0.4M NaCl is added. Thesuspension is mixed in a commercial blender for 30 seconds. The mixtureis centrifuged for 15 minutes at 17,000 Xg in the cold (4° C.). Thesupernatant is collected and ethanol is added to a concentration of 25%.This material is then centrifuged for 2 hours at 17,000 Xg (4° C.) andthe supernatant saved. Ethanol, at four times the volume of thesupernatant, is added and the material held overnight at 4° C.

2. Removal of Nucleic Acids

The material is centrifuged for 5 minutes at 2800 Xg (4° C.). Thesediment is collected and resuspended in Tris-MgSO₄ buffer at one-fourththe volume originally used to extract the paste. The composition of theTris buffer is as follows per liter of distilled water:

    ______________________________________                                        tris-hydroxymethylamino-methane (Sigma)                                                                6 gm                                                 MgSO.sub.4.7 H.sub.2 O  246 mg                                                thimerosal (Elanco)      50 mg                                                ______________________________________                                    

The pH is adjusted to 7.0±0.2 with concentrated hydrochloric acid.

Deoxyribonuclease I 1.5 mg (Sigma D-0876) and ribonuclease-A 0.75 mg(Sigma-Type 1-AS, R-5503) per 100 gm of original wet paste are added.The material is placed in a dialysis bag and incubated for 18 hours at37° C. versus 18 liters of Tris-MgSO₄ buffer.

3. Removal of Proteins

The material is further processed to remove protein components by addingan equal volume of phenol-acetate solution (135 ml of 10 percent (w/v)sodium acetate combined with 454 gms of phenol, AR grade). The materialsis then shaken for 30 minutes (4° C.), centrifuged for 15 minutes at17,000 Xg and the aqueous phase collected. Two additional phenolextractions are conducted followed by dialysis of the last aqueous phaseversus distilled water.

The material at this stage constitutes the bulk liquid capsularpolysaccharide (PRP) and is stored at -20° C. until further processing(Section D below).

4. Assessment of Quality of Polysaccharide

The quality of the bulk PRP is judged based on the analysis of a liquidsample that is removed. Ethanol is added (4 volumes), then CaCl₂ (finalconcentration of 0.02M) and the PRP is precipitated. The PRP issedimented by centrifugation and dried under vacuum over a dessicant. Athermogravimetric analysis (TGA) is used to determine the moisturecontent. Further analyses are calculated on a dry weight basis.

Criteria for acceptance include:

(a) analysis of ribose (Orcinol method): greater than 30 percent,

(b) analysis of protein (Lowry method): less than 1 percent,

(c) analysis of nucleic acids (U.V. adsorbance): less than 1 percent,and

(d) precipitation with specific immune sera by the(counterimmunoelectrophoresis method).

In addition, the molecular size is determined by suitable gel premeationchromatography. The polysaccharide is monitored for endotoxin by LimulusLysate testing and by rabbit pyrogenicity testing.

A typical lot has the following characteristics:

(a) Protein content, 0.5%

(b) Nucleic acid content, 0.35%

(c) Residual bovine antigens: No contamination of the purified PRP bybovine RNA-ase and DNA-ase used in preparation of the polysaccharide, asmesaured by radioimmunoassay.

(d) Endotoxin content was measured by the Limulus Amoebocyte to LysateAssay (LAL): 200 ng/mg PRP.

(e) Kd on CL-4B Sepharose: 0.30. A value of 0.30 corresponds to anapproximate molecular weight of 1,125,000 relative to dextran standards.

D. Preparation of Polysaccharide (PRP) Reagent

The polysaccharide is thus purified as a liquid, but calcium is not usedin the purification procedure. (Conventional polysaccharide purificationyields a calcium salt, but calcium can combine with the carbonate bufferused hereinbelow during conjugation to form a precipitate.)

The size of the polysaccharide is adjusted by heating at 100° C. for atime proportional to the degree of size change needed. Size of thepolysaccharide is adjusted so that less than 20% elutes from a CL-4BSepharose column in the void volume, and less than 20% elutes with a Kdgreater than 0.5. The principal fraction has a molecular weight of205,000 to 2,000,000 dalton.

EXAMPLE II Activation of PRP

A. This polysaccharide is cooled on an ice bath to 4° C. in a reactionvessel equipped with a magnetic stirrer. The initial volume of PRP atthe concentration of 25 mg/ml (20-30 mg/ml range) in distilled water isrecorded. Then sodium chloride is added to a concentration of 0.85%.

B. The pH of the polysaccharide is raised to 10.5-11.0 by addition of 1Nsodium hydroxide. (This range is chosen because at a lower pH there isless reaction with cyanogen bromide and at higher pH the polysaccharidebreaks down.)

C. Dry cyanogen bromide is dissolved in 0.005N sodium bicarbonate bufferof pH 10.5-11.0 and immediately (within 10 minutes of preparation) isadded to the reaction vessel in a proportion of 0.4 mg/mg PRP.

D. The pH of the mixture is adjusted to and maintained at 10.5-11.0 for6 minutes by addition of sodium hydroxide.

E. The pH is then dropped to 6.0 with 1N HCl. (Acid pH stabilizes theactivated sites created on the polysaccharide by cyanogen bromide.Lowering the pH further results in hydrolysis of the PRP.)

G. There is added an equal volume of saline, pH 6.0, prechilled to 4° C.

H. The cyanogen bromide-polysaccharide mixture is transferred to aconcentrator apparatus and concentrated to the initial volume recordedat step A.

I. Steps G and H are repeated at total of 10 times. Thus about 99.9% ofthe unconsumed cyanogen bromide is removed while the polysaccharideconcentration is maintained at 25 mg/ml. If the cyanogen bromide is notremoved, it reacts with the diphtheria toxoid used below.

EXAMPLE III Preparation of the D-AH Carrier

A. Commercial diphtheria toxoid (D) in distilled water is concentratedto 25 mg/ml over a membrane that retains molecules over 10,000 dalton ina positive pressure stirred concentrator.

B. A dry mixture of 8 mg adipic acid dihydrazide (ADH) per mg proteinand 0.75 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) per mgprotein are placed in a reaction vessel equipped with an efficientstirrer and a pH probe to permit monitoring and control of pH.

C. The diphtheria toxoid is then added to the reaction vessel making theproportion of the reactants:

diphtheria toxoid=25 mg protein/ml

ADH=8.0 mg/mg protein

EDAC=0.75 mg/mg protein

(The use of acetate buffer on sequential addition of ADH and EDACresults in a persistent flocculent precipitate. The reactants have asufficient buffering capacity of their own.)

D. The pH is immediately adjusted to 4.7 and maintained at 4.7±0.2 for aminimum of 2 hours.

1. The course of the chemical reaction can be followed on the recorderof the pH controller. If necessary, the reaction is allowed to proceedpast 2 hours until the pH is stable for at least 15 minutes, (i.e., 15minutes without needing HCl addition to control pH).

2. The amount of HCl consumed is recorded as a process check.

3. Temperature change in the reaction vessel is also monitored.

E. The reaction product is dialyzed at 4° C. against two changes ofsaline, a minimum 100 volumes and 8 hours per change.

F. It is then dialyzed against two changes of phosphate buffered saline,minimum volume, time and temperature as for step E.

G. The product (D-AH) is concentrated back to 25 mg/ml protein with apositive pressure apparatus and 10,000 MW membrane.

H. The concentrate is sterile filtered (0.2μ) and stored at 4° C.

Assay of a sample of a typical lot of such D-AH carrier showed a ratioof 38.3 microgram ADH/mg DT. Chromatography of that sample showed a Kdvalue on CL-4B Sepharose of 0.75 which corresponds to an approximatemolecular weight of 139,000 relative to protein standards.

EXAMPLE IV Formation of the Covalent Polysaccharide-diphtheria ToxoidConjugate (PRP-D)

A. The diphtheria toxoid adipic acid hydrazide carrier (D-AH) of ExampleIII, at a concentration of 25 mg/ml, is treated with sodium bicarbonateto a concentration of 0.5M and the pH is adjusted to 8.5. (Significantlylower salt concentrations result in the formation of a gel in the finalreaction mixture with activated polysaccharide.)

B. In a reaction vessel which can be sealed, an equal amount of thewashed PRP solution produced in Example II is added and the pH should bestable at 8.4-8.6. (If CNBr has not been removed, the pH will driftrapidly downward and a copious precipitate will form. This reactionoccurs even in the absence of polysaccharide. There should not be asignificant change of the physical appearance of the reactants oncombination.)

C. The reaction mixture is tumbled for 15-18 hours at 4° C.

D. The conjugate is purified by gel permeation chromatography onSephacryl-300 equilibrated in phosphate buffered saline to removeunreacted protein and low molecular weight material (less than 140,000dalton). (If the starting polysaccharide is too small it will not bepossible to separate the conjugate from free protein in this manner.)

E. Samples of this purified conjugate are removed for chemical analysis,described in the following section of this example.

F. Thimerosal is added to the purified conjugate to a concentration of1:10,000 and the product is stored at 4° C. until analysis.

G. The product is 0.2 micron sterile filtered. (If the polysaccharide isnot sized as described in Example I(D), i.e., if it is oversize, theresulting conjugate will not be filterable.)

ANALYSIS

Tests performed on the bulk concentrate of Example IV gave the followingresults:

(a) Ribose content: 156.5 microgram/ml.

(For the calculation of PRP values in Example V, a conversion factor of2 is used to calculate a nominal polysaccharide concentration based onthe empirically determined ribose concentration.)

(b) Protein content: 330 microgram/ml.

(c) Ribose/protein ratio: 0.47 (limits 0.25-0.75)

(d) Chromatographic analysis on Sepharose CL-4B gave the chromatographicprofile of FIG. 1. The solid line shows the curve for microgramprotein/ml, the broken line that for polysaccharide/ml.

(e) Kd (polysaccharide): 0.36

Determined by individual fraction ribose assay for PRP. A value of 0.36corresponds to an approximate molecular weight of 674,000 relative todextran standards.

(f) Kd (protein): 0.34

Determined by individual fraction Lowry assay for protein. The change inthe Kd value of the diphtheria toxoid from 0.75 to 0.34 shows that theconjugation of protein with polysaccharide forms a molecule which ischromatographically different from either raw material.

(g) Free Protein: Less than 5%.

Free protein represents derivatized diphtheria carrier protein which asnot been bound to PRP. It is determined by comparing the amount ofprotein that elutes in the position of a diphtheria toxoid samplerelative to the total eluted protein.

(h) Endotoxin Content: 1 ug/ml

Endotoxin was quantitated by the Limulus Amoebocyte to Lysate Assay(LAL). The endotoxin content amounts to 64 ng per 10 ug ribose humandose of PRP-D.

(i) Pyrogenicity:

The bulk concentrate meets the standards for pyrogenicity set forth in21 CFR 610.13(b) using a weight equivalent (human) dose of 0.15 ugribose per milliliter per kilogram body weight of rabbit.

(j) Polyacrylamide Gel Electrophoresis (PAGE):

PAGE analysis was performed to obtain supporting evidence of purity andcovalent bonding between polysaccharide and protein. While free carrierbands just over halfway into the rod gel, at approximately the positionof catalase (60,000 MW), the PRP-D conjugate was not able to enter thegel (prior to the position of thyroglobulin, 330,000 MW). PRP-D showed asingle band at the origin.

(k) Cyanogen Bromide:

While cyanogen bromide (CNBr) is used in the first steps beforepreparing a PRP-D conjugate, it is subsequently excluded from theproduct. Several steps of the process contribute to the reduction ofCNBr content. However, final purification of the vaccine by gelpermeation chromatography removes any contaminants below 100,000 MW.This purification precludes contamination with residual traces of freeCNBr or its degradation products.

EXAMPLE V PRP-D Immunogenicity Testing

This experiment was designed to show T-cell dependency as evidenced by:carrier dependency, carrier specificity, booster effect, and Ig classswitch. This experiment was carried out in two parts: (1) an initialpriming sequence of two injections; (2) a challenge sequence of twoinjections.

Materials:

1. PRP-D Bulk concentrate, Example 4.

2. Tetanus Toxoid (TT).

3. Diphtheria Toxoid (DT).

4. H. influenzae b capsular polysaccharide (PRP).

5. PRP/DT, a mixture of 20 ug PRP (10 ug ribose) and 20 ug DT.

6. PRP/DT-AH, a mixture of 20 ug PRP (10 ug ribose) and 20 ug DT-AH.

METHOD

All immunizations were administered subcutaneously without adjuvant in 1ml volumes. All doses containing PRP were adjusted to 10 ug ribose per 1ml dose. Unconjugated toxoid doses were adjusted to 20 ug protein per 1ml dose. A rotating schedule was used with immunizations spaced 14 daysapart. Each immunization was followed by a bleed 10 days later. Allpreparations were diluted in phosphate buffered saline containing 0.01%thimerosal. Aliquots were prepared as four dose vials, and stored at-20° C. Three rabbits were immunized in each group.

SEROLOGY

Sera were assayed by the solid phase radioimmunoassay (SPRIA) foranti-PRP, anti-DT-AH, anti-DT, and anti-TT as indicated. Antibodieslevels were quantitated as microgram IgG and IgM per ml.

    ______________________________________                                        Protocol:                                                                                                     SPRIA                                         Group  Primary  Secondary  PRP  DT-AH  DT   TT                                ______________________________________                                        1      PRP      PRP        +                                                  2      TT       PRP-D      +    +      +    +                                 3      DT       PRP-D      +    +      +    +                                 4      PRP/DT   PRP-DT     +           +                                      5      PRP/     PRP/DT-AH  +    +                                                    DT-AH                                                                  6      PRP-D    PRP-D      +    +      +                                      ______________________________________                                    

SCHEDULE

Rabbits were pre-bled and immunized according to group every 14 days.Each immunization was followed by a post-bleed 10 days later. The firsttwo injections were made with the primary immunogen, while the third andfourth injections were made with the secondary immunogen.

Anti-PRP response is demonstrated in FIG. 2, below. The mean level ofIgG antibody to Haemophilus influenzae b capsular polysaccharide in thesix experimental groups of rabbits (three animals per group) aregraphically illustrated. Groups are labelled on this figure inaccordance with the above protocol. Prebleed levels were less than 1μg/ml for all rabbits and are not shown in this figure for clarity.

Open bars represent the level of IgG following two successive injectionswith the primary immunogen. Solid bars show the IgG levels following thethird and fourth injections which were with the secondary or challengeimmunogens.

IgG responses were seen only following immunization with the PRP-Dconjugate vaccine. Priming the rabbits in group 3 with diphtheria toxoidaccelerated the response to PRP-D, while priming the tetanus toxoid(group 2) had no effect.

These results are included in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    ANTI-PRP RESPONSE                                                             Solid Phase Radioimmunoassay for IgM and IgH                                           PRP      Diphtheria Toxoid                                                                       Tetanus Toxoid                                             IgG*                                                                              IgM  IgG  IgM  IgG  IgM                                          __________________________________________________________________________    Group 1                                                                            Pre 0.0.sup.#                                                                         0    ND.sup.%  ND                                                     Post 1                                                                            0.33 ±                                                                         0                                                                         0.58                                                                      Post 2                                                                            0.8 ±                                                                          0                                                                         1.39                                                                      Post 3                                                                            0   0                                                                     Post 4                                                                            0   0                                                                Group 2                                                                            Pre 0   0    0    0    0.5 ±                                                                           0                                                                        0.87                                                   Post 1                                                                            0   0    0    0    25.1 ±                                                                          113.1 ±                                                               15.10                                                                              129.54                                            Post 2                                                                            0   0    0    0    112.00 ±                                                                        44.60 ±                                                               0    36.90                                             Post 3                                                                            4.82 ±                                                                         26.3 ±                                                                          0    0    112.00 ±                                                                        8.50 ±                                             2.59                                                                              45.55          0    14.72                                             Post 4                                                                            36.1 ±                                                                         74.57 ±                                                                         0    0    151.60 ±                                                                        2.00 ±                                             17.43                                                                             28.81          52.64                                                                              3.46                                         Group 3                                                                            Pre 0.2 ±                                                                          0    0    27.07 ±                                                                         0.8 ±                                                                           65.00 ±                                            0.35          46.88                                                                              1.39 112.58                                            Post 1                                                                            0.2 ±                                                                          0    0    0    0.8 ±                                                                           0                                                     0.35               1.39                                                   Post 2                                                                            0   14.6 ±                                                                          6.47 ±                                                                          0    0.67 ±                                                                          0                                                         25.29                                                                              5.98      0.59                                                   Post 3                                                                            19.4 ±                                                                         130.33 ±                                                                        10.63 ±                                                                         0    0.37 ±                                                                          0                                                     13.4                                                                              89.49                                                                              8.08      0.65                                                   Post 4                                                                            74.2 ±                                                                         82.63 ±                                                                         29.03 ±                                                                         0    1.30 ±                                                                          0                                                     76.73                                                                             5.16 26.59     2.25                                              Group 4                                                                            Pre 0   0    0    21.77 ±                                                                         ND                                                                       18.85                                                       Post 1                                                                            0   15.6 ±                                                                          2.67 ±                                                                          30.87 ±                                                          27.02                                                                              0.85 27.14                                                       Post 2                                                                            0   0    37.2 ±                                                                          26.2 ±                                                                10.94                                                                              24.30                                                       Post 3                                                                            0   0    210.00 ±                                                                        19.63 ±                                                               0    17.06                                                       Post 4                                                                            0   0    298.27 ±                                                                        0                                                                        207.70                                                      Group 5                                                                            Pre 0   0    ND                                                               Post 1                                                                            0   0                                                                     Post 2                                                                            0   0                                                                     Post 3                                                                            0   0                                                                     Post 4                                                                            0   0                                                                Group 6                                                                            Pre 0   0    0    0                                                           Post 1                                                                            6.23 ±                                                                         135.00 ±                                                                        0    0                                                               2.56                                                                              81.41                                                                 Post 2                                                                            54.2 ±                                                                         182.00 ±                                                                        0.93 ±                                                                          0                                                               14.73                                                                             0    1.62                                                             Post 3                                                                            57.6 ±                                                                         182.00 ±                                                                        3.05 ±                                                                          0                                                               22.91                                                                             0    4.31                                                             Post 4                                                                            39.6 ±                                                                         91.20 ±                                                                         5.95 ±                                                                          0                                                               25.17                                                                             0    8.41                                                        __________________________________________________________________________     *IgG and IgM are expressed as ug antibody per ml of serum. All value are      mean and standard error.                                                      #Ig levels below the sensitivity of the assay were tested as zero (0). If     levels higher than the range of the assay were assigned a value equal to      the upper limit of the assay.                                                 % ND = Not Done.                                                         

What is claimed is:
 1. A hapten prepared from capsular Haemophilusinfluenzae b polysaccharide, consisting of approximately equal parts ofribose, ribitol and phosphate, by heating until less than 20% is of amolecular size is below 200,000 dalton and less than 20% is of molecularsize greater than 2,000,000 dalton.